New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases

PloS One
Tao TuBin Yao

Abstract

Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32 (wild type PG8fn) ...Continue Reading

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Citations

Jan 1, 2017·Applied and Environmental Microbiology·Hong YangBin Yao
Aug 16, 2018·Applied Microbiology and Biotechnology·Hong YangBin Yao
Jan 21, 2021·Microbial Ecology·Luigimaria BorrusoCristina Giacoma
May 8, 2021·The Plant Journal : for Cell and Molecular Biology·Yang YangJiashu Cao

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Methods Mentioned

BETA
GTPase
PCR
electrophoresis
protein assay

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